Differences in genomic macrorestriction patterns of arabinose-positive (Burkholderia thailandensis) and arabinose-negative Burkholderia pseudomallei.

We reported previously two biochemically and antigenically distinct biotypes of Burkholderia pseudomallei. These two distinct biotypes could be distinguished by their ability to assimilate L-arabinose. Some B. pseudomallei isolated from soil samples could utilize this substrate (Ara+), whereas the other soil isolates and all clinical isolates could not (Ara-). Only the Ara isolates were virulent in animals and reacted with monoclonal antibody directed at the surface envelope, most likely the exopolysaccharide component. In the present study, pulsed-field gel electrophoresis was employed for karyotyping of these previously identified B. pseudomallei strains. We demonstrate here that the DNA macrorestriction pattern allows the differentiation between B. pseudomallei, which can assimilate L-arabinose, and the proposed B. thailandensis, which cannot do so. Bacterial strains from 80 melioidosis patients and 33 soil samples were examined by genomic DNA digestion with NcoI. Two major reproducible restriction patterns were observed. All clinical (Ara-) isolates and 9 Ara- soil isolates exhibited macrorestriction pattern I (MPI), while 24 soil isolates (Ara+) from central and northeastern Thailand displayed macrorestriction pattern II (MPII). The study here demonstrated pulsed-field gel electrophoresis to be a useful tool in epidemiological investigation possibly distinguishing virulent B. pseudomallei from avirulent B. thailandensis or even identifying closely related species of Burkholderia.     

Detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei and Burkholderia thailandensis by multiplex PCR

Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis may be differentiated from closely related species of Burkholderia mallei that causes glanders and non-pathogenic species of Burkholderia thailandensis by multiplex PCR. The multiplex PCR consists of primers that flank a 10-bp repetitive element in B. pseudomallei and B. mallei amplifying PCR fragment of varying sizes between 400-700 bp, a unique sequence in B. thailandensis amplifying a PCR fragment of 308 bp and the metalloprotease gene amplifying a PCR fragment of 245 bp in B. pseudomallei and B. thailandensis. The multiplex PCR not only can differentiate the three Burkholderia species but can also be used for epidemiological typing of B. pseudomallei and B. mallei strains.     

A possible pitfall in the identification of Burkholderia mallei using molecular identification systems based on the sequence of the flagellin fliC gene

Amotile Burkholderia mallei and motile Burkholderia pseudomallei display a high similarity with regard to phenotype and clinical syndromes, glanders and melioidosis. The aim of this study was to establish a fast and reliable molecular method for identification and differentiation. Despite amotility, the gene of the filament forming flagellin (fliC) could be completely sequenced in two B. mallei strains. Only one mutation was identified discriminating between B. mallei and B. pseudomallei. A polymerase chain reaction-restriction fragment length polymorphism assay was designed making use of the absence of an AvaII recognition site in B. mallei. All seven B. mallei, 12 out of 15 B. pseudomallei and 36 closely related apathogenic Burkholderia thailandensis strains were identified correctly. However, in three B. pseudomallei strains a point mutation at gene position 798 (G to C) disrupted the AvaII site. Therefore, molecular systems based on the fliC sequence can be used for a reliable proof of strains of the three species but not for the differentiation of B. mallei and B. pseudomallei isolates.     

Simultaneous detection of Fusarium asiaticum and Fusarium graminearum in wheat seeds using a real-time PCR method

Aims:  To develop a PCR-based method for quantitative detection of Fusarium asiaticum ( Fa ) and Fusarium graminearum ( Fg ) in wheat seeds.
Methods and Results:  Based on the sequences of the cyp51A gene, two primer pairs FaF + FaR and FgF + FgR were developed for the species-specific detection of Fa and Fg , respectively. To simultaneously detect these two phylogenetic species, a pair of primers FgaF + FgaR was developed based on the first and the second introns of β -tubulin gene. This primer pair amplified a 228-bp fragment only from Fa and Fg isolates, but not from 22 other Fusarium spp. and 13 other fungal species. A real-time PCR with this primer pair was able to quantify minute amounts of Fa and Fg DNA in wheat seeds rapidly.
Conclusions:  PCR primers designed based on the sequence of cyp51A or intron region of β -tubulin gene could allow differentiation of genetically related fungal species.
Significance and Impact of the Study:  The sensitive and quantitative detection method can be readily used in epidemiological studies and in assessing risk of Fusarium mycotoxin contamination in wheat samples.     

Flagellar glycosylation in Burkholderia pseudomallei and Burkholderia thailandensis

Glycosylation of proteins is known to impart novel physical properties and biological roles to proteins from both eukaryotes and prokaryotes. In this study, gel-based glycoproteomics were used to identify glycoproteins of the potential biothreat agent Burkholderia pseudomallei and the closely related but nonpathogenic B. thailandensis. Top-down and bottom-up mass spectrometry (MS) analyses identified that the flagellin proteins of both species were posttranslationally modified by novel glycans. Analysis of proteins from two strains of each species demonstrated that B. pseudomallei flagellin proteins were modified with a glycan with a mass of 291 Da, while B. thailandensis flagellin protein was modified with related glycans with a mass of 300 or 342 Da. Structural characterization of the B. thailandensis carbohydrate moiety suggests that it is an acetylated hexuronic acid. In addition, we have identified through mutagenesis a gene from the lipopolysaccharide (LPS) O-antigen biosynthetic cluster which is involved in flagellar glycosylation, and inactivation of this gene eliminates flagellar glycosylation and motility in B. pseudomallei. This is the first report to conclusively demonstrate the presence of a carbohydrate covalently linked to a protein in B. pseudomallei and B. thailandensis, and it suggests new avenues to explore in order to examine the marked differences in virulence between these two species.     

Identification of the causative agents of glanders and melioidosis by polymerase chain reaction

Burkholderia mallei and B. pseudomallei are causative agents of glanders and melioidosis, respectively, i.e. severe and fatal infection diseases of man and animal. The computer-based analysis of the 23S rRNA gene sites was used for selecting the primers. Two pairs of primers were chosen for the identification of B. mallei and Bpseudomallei. DNAs from 48 B. pseudomallei and 15 strains of B. mallei, unlike from other geterological bacteria, were positively amplified. Therefore, the method of polymerase chain reaction can be used in laboratory diagnosis of glanders and melioidosis.     

Differentiation of Salmonella enterica serotype gallinarum biotype pullorum from biotype gallinarum by analysis of phase 1 flagellin C gene (fliC)

Salmonella enterica serotype gallinarum biotype gallinarum and biotype pullorum are non-motile and pathogenic avian strains. Biotype gallinarum causes fowl typhoid and biotype pullorum is the cause of pullorum disease in chickens. The two biotypes could be differentiated based on biochemical characteristics. However, conventional culture and biochemical assays are time-consuming, laborious and need sterile laboratory practices. Although the two biotypes, gallinarum and pullorum are non-motile, they possess the phase 1 flagellin C gene. The variable regions of the flagellin C gene from 41 biotype pullorum and 52 biotype gallinarum were amplified by colony-PCR and analyzed by single strand conformational polymorphism (SSCP) method. Differences in SSCP electrophoretic patterns were confirmed by nucleotide sequencing. In addition, PCR-RFLP with Hinp1I was also successfully applied to differentiate the two biotypes. These results suggested that the variable regions of fliC could be used as a genetic marker to differentiate biotype gallinarum from biotype pullorum.     

Analysis of the presence ofprtR proteinase gene in natural isolates ofLactobacillus rhamnosus

The region of the prtR gene coding for the active site of PrtR proteinase was detected in natural isolates of lactobacilli, previously determined as Lactobacillus rhamnosus. This region was present in all L. rhamnosus strains with proteolytic activity. The PCR primers used were constructed on the basis of the sequence of the catalytic domain of the prtR proteinase gene. These primers generated in colony-PCR procedure specific 611 1-bp product with DNA from natural isolates of L. rhamnosus. No PCR amplifications using these primers were obtained for closely related bacteria of genus Lactobacillus, regardless of their proteolytic activity. In addition, these primers could be used singly or in multiplex PCR together with the Lactobacillus genus-specific primers. Compared with the other proteinases within the genus Lactobacillus (PrtP, PrtB and PrtH) which retained the activity in cell-free proteinase extracts, PrtR proteinase showed proteolytic activity only under in vivo conditions (whole cells of the producing strains).     

Variation in Flagellin Genes and Proteins of Burkholderia cepacia

The majority of isolates of Burkholderia cepacia, an important opportunistic pathogen associated with cystic fibrosis, can be classified into two types on the basis of flagellin protein size. Electron microscopic analysis indicates that the flagella of strains with the larger flagellin type (type I) are wider in diameter. Flagellin genes representative of both types were cloned and sequenced to design oligonucleotide primers for PCR amplification of the central variable domain of B. cepacia flagellin genes. PCR-restriction fragment length polymorphism analysis of amplified B. cepacia flagellin gene products from 16 strains enabled flagellin type classification on the basis of product size and revealed considerable differences in sequence, indicating that the flagellin gene is a useful biomarker for epidemiological and phylogenetic studies of this organism.Burkholderia cepacia (formerly Pseudomonas cepacia; a member of the rRNA group II pseudomonads) has emerged as an increasingly important opportunistic pathogen, particularly in relation to patients suffering from cystic fibrosis (CF) (15). Acquisition of B. cepacia, often occurring after lengthy colonization with Pseudomonas aeruginosa, can lead to the rapid deterioration or death of CF patients, and this organism appears to be transmissible between patients (14). There is considerable evidence that some strains of B. cepacia are more virulent than others and that the outcome of colonization by a particular strain can vary from rapidly fatal septicemia to maintenance of stable respiratory function (16). A number of factors have been implicated in the greater virulence of some strains. These include adhesion to respiratory mucin (31, 32) and the presence of cable pili (33).Motility in B. cepacia is by means of polar flagella. Flagella, each consisting of a flagellin filament, hook, and basal body, have been implicated as invasive virulence factors for a number of bacteria (28), including P. aeruginosa (11). Unlike P. aeruginosa, which appears to sit in microcolonies in the viscid mucus, leading to progressive lung damage with episodes of acute debilitating exacerbation, some strains of B. cepacia cause rapidly fatal pneumonia in CF patients (15), suggesting that they may have the ability to move through the mucus. Because of their location on the outside of bacterial cells, flagellins have been targeted in vaccine design. Brett et al. (4) demonstrated that flagellin-specific antisera were capable of protecting diabetic rats from challenge with strains of Burkholderia pseudomallei (another member of rRNA group II). In a recent study, an O-polysaccharide moiety of B. pseudomallei was covalently linked to the flagellin protein from the same strain. O-polysaccharide–flagellin conjugates elicited a high-level immunoglobulin G response capable of protecting diabetic rats from challenge with a heterologous strain of B. pseudomallei (5).Two distinct flagellin protein molecular mass groups in B. cepacia have been reported by Montie and Stover (23). In this previous study, type I flagellins were reported as having a molecular mass of 31 kDa while the molecular mass of type II flagellins was reported as 44 to 46 kDa. This early study, using a limited number of isolates, suggested that with regard to flagellin, B. cepacia is analogous to another CF pathogen, P. aeruginosa, in which two flagellin antigenic types distinguishable by protein or gene size are found (43). Several representatives of the heterologous a-type and homologous b-type fliC loci of P. aeruginosa (encoding flagellins) have been sequenced (37). In addition, PCR amplification of flagellin genes coupled with restriction fragment length polymorphism (RFLP) analysis can be used as a method for differentiating between clinical isolates of P. aeruginosa (7, 43). In this paper we report the development of a similar approach to the study of populations of B. cepacia and discuss the divergence of a highly variable gene, the flagellin gene (fliC), within populations of B. cepacia.     

Rapid PCR-based test for identifying Candida albicans by using primers derived from the pH-regulated KER1 gene

A PCR-based method in combination with a simple, reliable and inexpensive DNA extraction procedure for rapid detection of Candida albicans clinical isolates is described here. The extraction protocol is based on a combination of chemical (NaOH and detergents) and physical (boiling) treatments, thus avoiding many of the problems inherent in the currently available DNA extraction protocols (basically the use of expensive and/or toxic chemical reagents), and may be useful for daily clinical routine. The PCR-based system described here uses a single pair of primers (SC1F and SC1R) deduced from the C. albicans-specific KER1 gene sequence. These primers amplify a 670-bp fragment of the KER1 gene. All the clinical C. albicans isolates generated the expected 670-bp amplicon. Other non-albicans Candida species, including the azole-resistant C. krusei and C. glabrata, and the very closely related C. dubliniensis, failed to amplify any DNA fragment. The PCR results reported here suggest that amplification with SC1F and SC1R primers is species specific and, consequently, may be useful for specifically identifying C. albicans strains.     

Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower

Alternaria helianthi is an important seed-borne pathogenic fungus responsible for blight disease in sunflower. The current detection methods, which are based on culture and morphological identification, are time-consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on the sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers—ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of A. helianthi. The specificity of the primer pairs AhN1F and AhN1R designed was verified by PCR analysis of DNA from 18 Alternaria helianthi strains isolated from India, 14 non-target Alternaria spp. and 11 fungal isolates of other genera. A single amplification product of 357-bp was detected from DNA of A. helianthi isolates. No cross-reaction was observed with any of the other isolates tested. The detection limit of the PCR method was of 10?pg from template DNA. The primers could also detect the pathogen in infected sunflower seed. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of A. helianthi. This is the first report of an A. helianthi-specific primer set.     

Specific detection of Salmonella spp. by multiplex polymerase chain reaction.

Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.     

Isolation and identification of Burkholderia pseudomallei from soil using selective culture techniques and the polymerase chain reaction

An environmental soil survey to detect Burkholderia pseudomallei was performed during the dry and wet seasons in Darwin, Northern Territory, Australia. Soil was sampled at regular intervals during a 15-month period at different depths from areas which were representative of the local, soil environment. Selective culture techniques using Ashdown's and Galimand and Dodin's methods and the polymerase chain reaction (PCR) using specific 16S rRNA primers were used to detect and identify the organism and determine its distribution within the soil stratum over the change in seasons. Results showed that Ashdown's method gave higher isolation rates in the dry season, and Galimand and Dodin's method gave higher isolation rates during the wet season. PCR of the soil enrichment proved to be a more sensitive method than culture and was also a useful confirmatory test in determining the identification of isolates where biochemical tests gave inconsistent results. The PCR primers were specific and able to detect 10¹ cfu g^-1 soil and 10⁴ cfu g^-1 of soil using Ashdown's enrichment broth and Galimand and Dodin's broth, respectively. Overall the isolation of B. pseudomallei was greatest during the dry season and at the higher and lower soil depths, which is contradictory to epidemiological evidence that melioidosis occurs primarily during the wet season among patients exposed to contaminated surface soil and water.     

Differential detection of vibrios pathogenic to shrimp by multiplex PCR

The research was focused on the multiplex polymerase chain reaction (PCR) differential detection of shrimp pathogens Vibrio harveyi, Vibrio campbellii and isolates from a variant strain of Vibrio (referred to as Philippine Vibrio isolates in this study) exhibiting characteristics distinct from these two species. Sequence alignment of the hemolysin gene from type strains Vibrio harveyi (NBRC 15634) and Vibrio campbellii (NBRC 15631), as well as 10 variant Philippine Vibrio isolates, was performed in order to design a set of hemolysin-targeted primers for the specific detection of the Philippine Vibrio isolates. Primer PNhemo amplified a 320-bp hemolysin gene fragment of the Philippine Vibrio isolates in PCR using 65 degrees C annealing temperature, but did not amplify the target gene fragment in type strains V. harveyi and V. campbellii. Another new primer (VcatoxR) targeting the toxR gene was designed for the specific detection of type strain V. campbellii under stringent 65 degrees C annealing temperature. PCR using VcatoxR primer resulted in the specific amplification of a 245-bp V. campbellii toxR fragment. The simultaneous use of three primer sets in PCR, including PNhemo and VcatoxR (the two new primers designed in this study), and a primer VhtoxR (previously reported for the specific detection of V. harveyi), resulted in differential profiles with 390-bp, 245-bp, and 320-bp amplicons for V. harveyi, V. campbellii, and variant Philippine Vibrio isolates, respectively. Presence of all three types of Vibrio shrimp pathogens in the sample could be detected with a multiplex PCR profile containing all the expected size amplicons.     

Discrimination of Six Pratylenchus Species Using PCR and Species-Specific Primers

A PCR-based assay for identification of six species of Pratylenchus common in California is described. In this assay, five forward species-specific primers were designed from the internal variable portion of the D3 expansion region of the 26S rDNA and were each used with a single, common reverse primer. The optimized species-specific primers produced unique amplicons from their respective target and did not amplify DNA from other Pratylenchus species. With this assay we were able to identify single females to species level. This method obviates the need for subsequent RFLP or sequence analysis of the PCR product and can be used as a rapid diagnostic tool in epidemiological and management studies.     

Deletion commonly found in Waxy gene of Japanese and Korean cultivars of Job’s tears (Coix lacryma-jobi L.)

We isolated the entire sequence of the coding region of Waxy gene of a non-waxy accession of Job??s tears (Coix lacryma-jobi) by PCR-based methods. We also compared the entire sequences of the gene between two non-waxy accessions and three waxy cultivars and found a 275-bp deletion in the coding region (exons 10?C11) of this gene specific to waxy cultivars. We showed by PCR genotyping that this deletion is commonly found in Japanese and Korean cultivars and confirmed that this deletion resulted in lack of Wx protein. We also confirmed that this polymorphism of the gene co-segregates with phenotypes in endosperm and pollen. These results suggest that this PCR-based marker will be useful in breeding of Job??s tears and that genetic information obtained in other grass species will be also useful in genetics and breeding of Job??s tears.     

Characterization of a spirochaete isolated from a case of bovine digital dermatitis

AIMS: The aim of the study was to characterize a spirochaete isolated from the lesions of a cow with digital dermatitis (DD). METHODS AND RESULTS: The characterization was on the basis of its light and electron microscopic appearance, enzymic profile and DNA sequence analysis of its flagellin and 16S rRNA genes. The spirochaete was 6-8-microm long and 0.2-0.3 microm in diameter, and possessed seven to eight periplasmic flagella, with three to five helical turns. The enzymic profile of the bacterium resembles, but is not identical to that of Treponema brennaborense. Its flagellin gene sequence was identical to that of Treponema phagedenis but distinct from that of an ovine spirochaete. Analysis of a 1477-bp region of the 16S rRNA genes indicated that this is a Treponema species and that it is indistinguishable from some isolates made from cases of bovine DD in the United States. Finally, electron microscopy revealed the presence of myovirus-like bacteriophage particles in all cultures of the treponeme examined. CONCLUSIONS: The spirochaete isolate was identified as a Treponema species closely related to some isolates from the United States (by 16S rDNA) and to T. phagedenis (by flagellin gene sequence) and is associated with bacteriophage particles. SIGNIFICANCE AND IMPACT OF THE STUDY: The fact that the isolates with the same or very similar 16S rDNA sequences have been obtained from cases of bovine DD in cattle in different countries at different times, lends further support to the hypothesis that treponemes play a role in the pathogenesis of this disease.     

Development of silverleaf assay, protein and nucleic acid-based diagnostic techniques for the quick and reliable detection and monitoring of biotype B of the whitefly, Bemisia tabaci (Gennadius)

The aim of this study was to develop and optimize silverleaf bioassay, esterase analysis and PCR-based techniques to distinguish quickly and reliably biotype B of the whitefly, Bemisia tabaci (Gennadius), from Indian indigenous biotypes. Zucchini and squash readily develop silverleaf symptoms upon feeding by the B biotype, but they are not readily available in Indian markets. A local pumpkin variety 'Big' was, therefore, used in silverleaf assay, which developed symptoms similar to those on zucchini and squash and can be used reliably to detect B biotype. Analysis of non-specific esterases of B and the indigenous biotypes indicated both quantitative and qualitative differences in esterase patterns. Two high molecular weight bands were unique to B biotype and they occurred in abundance. These esterases were used to develop quick and field-based novel detection methods for differentiating B from the indigenous biotypes. Development of these simple and cost-effective protocols has wider application as they can be potentially used to identify other agricultural pests. Mitochondrial cytochrome oxidase I gene sequences and randomly amplified polymorphic DNA (RAPD) polymorphisms, generated using the primer OpB11, were also found useful for detecting B. tabaci biotypes. A B biotype-specific RAPD band of 800 bp was sequenced, which was used to a develop sequence characterized amplified region (SCAR) marker. The SCAR marker involved the development of B biotype-specific primers that amplified 550 bp PCR products only from B biotype genomic DNA. Silverleaf assay, esterases, RAPDs or a SCAR marker were used in combination to analyse whitefly samples collected from selected locations in India, and it was found that any of these techniques can be used singly or in combination to detect B biotype reliably. The B biotype was found in southern parts of India but not in the north in 2004-06.